Efficient display of two enzymes on filamentous phage using an improved signal sequence

Directed protein-evolution strategies generally make use of a link between a protein and the encoding DNA. In phage-display technology, this link is provided by fusion of the protein with a coat protein that is incorporated into the phage particle containing the DNA. Optimization of this link can be achieved by adjusting the signal sequence of the fusion. In a previous study, directed evolution of signal sequences for optimal display of the Taq DNA polymerase I Stoffel fragment on phage yielded signal peptides with a 50-fold higher incorporation of fusion proteins in phage particles. In this article, we show that for one of the selected signal sequences, improved display on phage can be generalized to other proteins, such as adenylate cyclases from Escherichia coli and Bordetella pertussis, and that this is highly dependent on short sequences at the C-terminus of the signal peptide. Further, the display of two enzymes on phage has been achieved and may provide a strategy for directing coevolution of the two proteins. These findings should be useful for display of large and cytoplasmic proteins on filamentous phage.     

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2–P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352–356 (P7–P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7–P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of “designer serpins” with novel reactivity and/or specificity.     

Biotin-tagged cDNA expression libraries displayed on lambda phage: a new tool for the selection of natural protein ligands

cDNA expression libraries displayed on lambda phage have been successfully employed to identify partners involved in antibody–antigen, protein– protein and DNA–protein interactions and represent a novel approach to functional genomics. However, as in all other cDNA expression libraries based on fusion to a carrier polypeptide, a major issue of this system is the absence of control over the translation frame of the cDNA. As a consequence, a large number of clones will contain lambda D/cDNA fusions, resulting in the foreign sequence being translated on alternative reading frames. Thus, many phage will not display natural proteins, but could be selected, as they mimic the binding properties of the real ligand, and will hence interfere with the selection outcome. Here we describe a novel lambda vector for display of exogenous peptides at the C-terminus of the capsid D protein. In this vector, translation of fusion peptides in the correct reading frame allows efficient in vivo biotinylation of the chimeric phage during amplification. Using this vector system we constructed three libraries from human hepatoma cells, mouse hepatocytic MMH cells and from human brain. Clones containing open reading frames (ORFs) were rapidly selected by streptavidin affinity chromatography, leading to biological repertoires highly enriched in natural polypeptides. We compared the selection outcome of two independent experiments performed using an anti-GAP-43 monoclonal antibody on the human brain cDNA library before and after ORF enrichment. A significant increase in the efficiency of identification of natural target peptides with very little background of false-positive clones was observed in the latter case.     

Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display

Even proteins that fold well in bacteria are frequently displayed poorly on filamentous phages. Low protein presentation on phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in phage assembly, we tested the display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved phage display from tenfold enrichment to >1,000-fold enrichment per phage display selection round. We named this method 'SRP phage display' and envision broad applicability, especially when displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.     

A filamentous phage display system for N-linked glycoproteins

We have developed a filamentous phage display system for the detection of asparagine-linked glycoproteins in Escherichia coli that carry a plasmid encoding the protein glycosylation locus (pgl) from Campylobacter jejuni. In our assay, fusion of target glycoproteins to the minor phage coat protein g3p results in the display of glycans on phage. The glyco-epitope displayed on phage is the product of biosynthetic enzymes encoded by the C. jejuni pgl pathway and minimally requires three essential factors: a pathway for oligosaccharide biosynthesis, a functional oligosaccharyltransferase, and an acceptor protein with a D/E-X₁-N-X₂-S/T motif. Glycosylated phages could be recovered by lectin chromatography with enrichment factors as high as 2 × 10⁵ per round of panning and these enriched phages retained their infectivity after panning. Using this assay, we show that desired glyco-phenotypes can be reliably selected by panning phage-displayed glycoprotein libraries on lectins that are specific for the glycan. For instance, we used our phage selection to identify permissible residues in the −2 position of the bacterial consensus acceptor site sequence. Taken together, our results demonstrate that a genotype–phenotype link can be established between the phage-associated glyco-epitope and the phagemid-encoded genes for any of the three essential components of the glycosylation process. Thus, we anticipate that our phage display system can be used to isolate interesting variants in any step of the glycosylation process, thereby making it an invaluable tool for genetic analysis of protein glycosylation and for glycoengineering in E. coli cells.     

Novel helper phage design: intergenic region affects the assembly of bacteriophages and the size of antibody libraries

Abstract Phagemid vectors for display of proteins/peptides on the surface of filamentous phage utilize a plasmid genome carrying the phage origin of replication, along with the gene fused to a fragment of gene III. Generation of phage particles displaying the fusion protein also requires superinfection of the host bacterium with a helper virus. We describe here the construction of a new gene III mutant of M13 KO7 bacteriophage and compare its ability to act as helper phage with two mutants derived from Fd tet (fKN 16 and fCA 55). Furthermore, we investigate their capability to act as helper phages in SAP selection, where non-infectious helper phage, expressing antibody fragments but not protein 3, can still infect by first reacting with a soluble antigen-protein 3 fusion protein. Gene III mutants were found to be non-infectious, and high titers of infective particles were obtained only when the helper phage was grown in cells harbouring a gene III-containing plasmid. An amplification of the phage titer of 10⁶× was achieved in M13-derived phages, when used for the selection of specific antibody fragments.     

大分子量重组蛋白在丝状噬菌体表面的展示表达及其与小分子相互作用的初步研究

利用pHEN1KM13噬菌粒系统表达融合蛋白,进而确定大分子量重组蛋白在丝状噬菌体表达的部位及其表达后的生物活性。通过蛋白酶切处理前后噬菌体侵染细菌能力的变化快速地检测大分子蛋白质能否在噬菌体表面展示表达;比较了谷胱甘肽S转移酶及其与三个不同长度连接臂融合的外源蛋白在噬菌体表面的表达和组装,确定了不大于40kD的重组蛋白分子能展示表达在丝状噬菌体表面;并利用已知的小分子化合物与蛋白质的相互作用证明了组装在噬菌体表面的谷胱甘肽S转移酶重组蛋白仍保持其天然的结合活性,为利用噬菌体展示系统研究蛋白质与小分子化合物的相互作用建立了基础。     

A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.     

A Phage Display Technique for a Fast, Sensitive, and Systematic Investigation of Protein–Protein Interactions

Phage display is a technique in which a foreign protein or peptide is presented at the surface of a (filamentous) bacteriophage. This system, developed by Smith [(1985), Science 228, 1315–1317], was originally used to create large libraries of antibodies for the purpose of selecting those that strongly bound a particular antigen. More recently it was also employed to present peptides, domains of proteins, or intact proteins at the surface of phages, again to identify high-affinity interactions with ligands. Here we want to illustrate the use of phage display, in combination with PCR saturation mutagenesis, for the study of protein–protein interactions. Rather than selecting for mutants having high affinity, we systematically investigate the binding of every variant with its natural ligand. Via a modified ELISA we can calculate a relative affinity. As a model system we chose to display thymosin β4 on the phage surface in order to study its interaction with actin.     

Rapid antibody selection by mRNA display on a microfluidic chip

In vitro antibody-display technologies are powerful approaches for isolating monoclonal antibodies from recombinant antibody libraries. However, these display techniques require several rounds of affinity selection which is time-consuming. Here, we combined mRNA display with a microfluidic system for in vitro selection and evolution of antibodies and achieved ultrahigh enrichment efficiency of 10⁶- to 10⁸-fold per round. After only one or two rounds of selection, antibodies with high affinity and specificity were obtained from naïve and randomized single-chain Fv libraries of ~10¹² molecules. Furthermore, we confirmed that not only protein–protein (antigen–antibody) interactions, but also protein–DNA and protein–drug interactions were selected with ultrahigh efficiencies. This method will facilitate high-throughput preparation of antibodies and identification of protein interactions in proteomic and therapeutic fields.     

Identification of a collagen-binding protein from Necator americanus by using a cDNA-expression phage display library.

A phage display library was made starting from a cDNA library from the hematophagous human parasite Necator americanus. The cDNA library was transferred by polymerase chain reaction (PCR) cloning into phage display vectors (phagemids), using specially designed primers such that proteins would be expressed as fusions with the C-terminal part of the phage coat protein pVI. The vectors used are multicloning site variants of the original pDONG vectors described by Jespers et al. (1995). Electroporation of the ligation mixtures into electrocompetent Escherichia coli TGI cells yielded 3 x 10(8) pG6A, 1.9 x 10(8) pG6B, and 1 x 10(8) pG6C transfectants for N. americanus. The final libraries consisted of a mix of equal numbers of insert-containing phages from the A, B, and C libraries. Selection of phages for binding to human collagen was performed. Four rounds of panning on human collagens I and III resulted in a significant enrichment of collagen-binding phages from the N. americanus libraries. PCR analysis revealed various insert lengths; however, sequence determination indicated that all phages contained the same protein, albeit with different poly-A tail lengths. The encoded protein itself is a 135-amino acid protein (15 kDa), with no apparent homology to any other known protein. Next the protein was recloned into E. coli using the pET-15b-vector. Upon isopropyl-1-thio-beta-D-galactopyranoside induction, the recombinant protein, rNecH1, could be recovered by urea treatment from inclusion bodies. The rNecH1 protein binds to different collagens: human I > rat I > human III = calf skin I in a specific, dose-dependent, and saturable manner.     

Catalytic Turnover-Based Phage Selection for Engineering the Substrate Specificity of Sfp Phosphopantetheinyl Transferase

We report a high-throughput phage selection method to identify mutants of Sfp phosphopantetheinyl transferase with altered substrate specificities from a large library of the Sfp enzyme. In this method, Sfp and its peptide substrates are co-displayed on the M13 phage surface as fusions to the phage capsid protein pIII. Phage-displayed Sfp mutants that are active with biotin-conjugated coenzyme A (CoA) analogues would covalently transfer biotin to the peptide substrates anchored on the same phage particle. Affinity selection for biotin-labeled phages would enrich Sfp mutants that recognize CoA analogues for carrier protein modification. We used this method to successfully change the substrate specificity of Sfp and identified mutant enzymes with more than 300-fold increase in catalytic efficiency with 3′-dephospho CoA as the substrate. The method we developed in this study provides a useful platform to display enzymes and their peptide substrates on the phage surface and directly couples phage selection with enzyme catalysis. We envision this method to be applied to engineering the catalytic activities of other protein posttranslational modification enzymes.     

Direct measurement via phage titre of the dissociation constants in solution of fusion phage-substrate complexes.

Studies of interactions between filamentous fusion phage particles and protein or nucleic acid molecules have gained increasing importance with recent successes of screening techniques based upon random phage display libraries (biopanning). Since a number of different phage are usually obtained by biopanning, it is useful to compare quantitatively the binding affinities of individual phage for the substrate used for selection. A procedure is described for determination of relative dissociation constants (KdRel) between filamentous phage carrying peptide fusions to the coat protein gpIII and substrates in solution. This novel method is based on the measurement of phage titres. Phage selected from a random fusion phage library for binding to a monoclonal antibody or a viral structural protein exhibited KdRel values in the nanomolar and micromolar ranges for their respective substrates, thus validating the method over a wide range of binding affinities.     

Complete Genomic Sequence of Bacteriophage φEcoM-GJ1, a Novel Phage That Has Myovirus Morphology and a Podovirus-Like RNA Polymerase

The complete genome of EcoM-GJ1, a lytic phage that attacks porcine enterotoxigenic Escherichia coli of serotype O149:H10:F4, was sequenced and analyzed. The morphology of the phage and the identity of the structural proteins were also determined. The genome consisted of 52,975 bp with a G+C content of 44% and was terminally redundant and circularly permuted. Seventy-five potential open reading frames (ORFs) were identified and annotated, but only 29 possessed homologs. The proteins of five ORFs showed homology with proteins of phages of the family Myoviridae, nine with proteins of phages of the family Podoviridae, and six with proteins of phages of the family Siphoviridae. ORF 1 encoded a T7-like single-subunit RNA polymerase and was preceded by a putative E. coli σ⁷⁰-like promoter. Nine putative phage promoters were detected throughout the genome. The genome included a tRNA gene of 95 bp that had a putative 18-bp intron. The phage morphology was typical of phages of the family Myoviridae, with an icosahedral head, a neck, and a long contractile tail with tail fibers. The analysis shows that EcoM-GJ1 is unique, having the morphology of the Myoviridae, a gene for RNA polymerase, which is characteristic of phages of the T7 group of the Podoviridae, and several genes that encode proteins with homology to proteins of phages of the family Siphoviridae.     

Identification and molecular analysis of a multigene family encoding calliphorin, the major larval serum protein of Calliphora vicina

A library of Calliphora vicina genomic DNA was constructed in the λEMBL3 vector and screened for recombinant phages containing chromosomal segments encoding calliphorin, the major larval serum protein (LSP) of Calliphora. A large series of recombinants hybridizing with in vitro labelled poly(A)⁺ RNA from Calliphora larval fat bodies and with specific probes derived from the LSP-1 genes of Drosophila melanogaster was isolated. Five of these phages, chosen at random, were shown by hybrid selection to retain calliphorin mRNA specifically. Eleven calliphorin mRNA-homologous regions were located on restriction maps of these phages by hybridization with 5' end-labelled poly(A)⁺ RNA from Calliphora larval fat bodies. Each phage contains at least two calliphorin genes arranged in direct repeat orientation and seperated by 3.5–5 kb intergenic regions. The genes display similar but not identical restriction patterns. Filter hybridization and heteroduplex analysis indicate that they share a detectable homology with the LSP-1β gene of D. melanogaster. Whole genome Southern analysis showed that these genes belong to a large family of closely related calliphorin genes which were found by in situ hybridization to polytene chromosomes of trichogen cells to be clustered in region 4a of chromosome 2 of Calliphora vicina.     

A human scFv antibody generation pipeline for proteome research

The functional decryption of the human proteome is the challenge which follows the sequencing of the human genome. Specific binders to every human protein are key reagents for this purpose. In vitro antibody selection using phage display offers one possible solution that can meet the demand for 25,000 or more antibodies, but needs substantial standardisation and minimalisation. To evaluate this potential, three human, naive antibody gene libraries (HAL4/7/8) were constructed and a standardised antibody selection pipeline was set up. The quality of the libraries and the selection pipeline was validated with 110 antigens, including human, other mammalian, fungal or bacterial proteins, viruses or haptens. Furthermore, the abundance of VH, kappa and lambda subfamilies during library cloning and the E. coli based phage display system on library packaging and the selection of scFvs was evaluated from the analysis of 435 individual antibodies, resulting in the first comprehensive comparison of V gene subfamily use for all steps of an antibody phage display pipeline. Further, a compatible cassette vector set for E. coli and mammalian expression of antibody fragments is described, allowing in vivo biotinylation, enzyme fusion and Fc fusion.     

By-passing selection: direct screening for antibody–antigen interactions using protein arrays

We have developed a system to identify highly specific antibody–antigen interactions by protein array screening. This removes the need for selection using animal immunisation or in vitro techniques such as phage or ribosome display. We screened an array of 27 648 human foetal brain proteins with 12 well-expressed antibody fragments that had not previously been exposed to any antigen. Four highly specific antibody–antigen pairs were identified, including three antibodies that bind proteins of unknown function. The target proteins were expressed at a very low copy number on the array, emphasising the unbiased nature of the screen. The specificity and sensitivity of binding demonstrates that this ‘naive’ screening approach could be applied to the high throughput isolation of specific antibodies against many different targets in the human proteome.     

Isolation and Characterization of the New Mosaic Filamentous Phage VFJ Φ of Vibrio cholerae

Filamentous phages have distinguished roles in conferring many pathogenicity and survival related features to Gram-negative bacteria including the medically important Vibrio cholerae, which carries factors such as cholera toxin on phages. A novel filamentous phage, designated VFJΦ, was isolated in this study from an ampicillin and kanamycin-resistant O139 serogroup V. cholerae strain ICDC-4470. The genome of VFJΦ is 8555 nucleotides long, including 12 predicted open reading frames (ORFs), which are organized in a modular structure. VFJΦ was found to be a mosaic of two groups of V. cholerae phages. A large part of the genome is highly similar to that of the fs2 phage, and the remaining 700 bp is homologous to VEJ and VCYΦ. This 700 bp region gave VFJΦ several characteristics that are not found in fs2 and other filamentous phages. In its native host ICDC-4470 and newly-infected strain N16961, VFJΦ was found to exist as a plasmid but did not integrate into the host chromosome. It showed a relatively wide host range but did not infect the classical biotype O1 V. cholerae strains. After infection, the host strains exhibited obvious inhibition of both growth and flagellum formation and had acquired a low level of ampicillin resistance and a high level of kanamycin resistance. The antibiotic resistances were not directly conferred to the hosts by phage-encoded genes and were not related to penicillinase. The discovery of VFJΦ updates our understanding of filamentous phages as well as the evolution and classification of V. cholerae filamentous phage, and the study provides new information on the interaction between phages and their host bacteria.